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Recombinant Human Arginase-1
Arginase-1 (ARG-1) is a manganese-containing enzyme in the ureohydrolase family that catalyzes the hydrolysis of L-arginine into L-ornithine and urea (step 5 of the urea cycle). The type I isoform, discussed here, is expresssed predominantly in the liver and acts as a urea cycle component (1). Arg-1 is also a critical regulator of nitric oxide synthesis and vascular function with implications in many human diseases such as vascular disease, pulmonary disease, cancer, and has contributions to immune cell function (3). The activity of Arg is very specific and because of this specificity, modifications of the substrate structure and/or the stereochemistry severely lowers the kinetic activity. There is an autosomal recessive inherited disorder of arginase deficiency called argininemia, which is the most rare of all heritable defects in ureagenesis. Argininemia is characterized by excess L-arginine in the blood leading to hyperammonemia and also lowered arginase activity in the hepatic cells. Arg-1 definiciency presents with symptoms of seizures, vomiting, irritability, lethargy, intellectual impairment, behavior disturbances, and intermittent ataxia, to name a few (2). However, unlike other urea cycle disorders, Arg-1 deficiency does not entirely prevent ureagenesis. This is due to the increased Arg-II activity in the kidneys in individuals with arg-I deficiency. The enzymes in the kidneys will then catalyze ureagenesis, compensating for the decrease in arg-I activity in the liver. Due to this alternative method of urea synthesis, subjects with arg-I deficiency have longer lifetimes than those with other urea cycle defects. Uses of rhArg-1 Anti-Proliferative Activity Currently under clinical investigation, rhArg has shown anti-prolieferative acitivty in both melanoma and prostate cancer cells that are deficient in Ornithine Carbamoyl Transferase (OTC) due to the effects of arginine deprivation in pre-clinical evaluation. Cells deficient in OTC were used because normal cells can synthesize arginine from intracellular ornithine via OTC. This result was seen in vivo and in vitro (4). Multiple other papers support these results. Rh-Arg-I induced caspase-dependent apoptosis in non-Hodgkins lymphoma cells Researchers at Fudan University in Shanghai, China have recently used rh-Arg-I as a new experimental therapeutic for non-Hodgkins lymphoma via arginine deprivation. It was seen to have the ability to induce remarkable growth inhibition, cell cycle arrest, and caspase-dependent apoptosis. They also found that rh-arg treatment resulted in the appearance of autophagosomes and upregulation of microtubule-associated protein light chain 3 II, indicating that rhArg induced autophagy in lymphoma cells. Blocking autophagy using pharmocological inhibitors or genetic approaches (siRNA) enhanced the apoptotic effect of rhArg. These results demonstrated that rhArg has potent anti-lymphoma activity and that this activity can be enhanced by combination with autophagic inhibitors, both of which hold promising potential for novel therapeutics in non-Hodgkins lymphoma treatment. Uses in Research (general) Recombinant DNA is used to identify genes, map and sequence them, and determine their function. Recombinant DNA probes are used to evaluate expression levels of genes within cells. Construct Used to Express the Protein: pET30a(+)/ArgC Plasmid pET30a(+)/ArgC plasmid, containing a pET30(+) backbone and the human arginase gene (containing a non-coding sequence), was used to transform competent BL21 (DE3) E. coli cells. Host Cell: E. coli E. coli BL21 is used as a host cell with the plasmid vector described above. This particular strain was used because it has an RNA polymerase that very effectively transcribes the gene of interest. Protein Purification The plasmid was used to transform competent BL21 E. coli cells on LB plates containing kanamycin. Single colonies were picked and transferred into LB media. These cells were then fermented at 37 degrees C at 250 rpm. IPTG was then added to induce rhArg expression. rhARG was purified using nickel column. The specific activity of rhArg was approximately 200IU/mL and the purity is above 95%. One international unit of rhArg is the amount of enzyme that produces 1 micromolar urea per minute at 30 degrees C, pH 8.5 (5). References: 1. GeneCopoeia ProteoXpres Recombinant Proteins http://www.genecopoeia.com/product/recombinant-proteins/?gclid=CPfOxaW58bkCFYmf4Aodiy0AgQ 2. Medscape: Hyperammonemia http://emedicine.medscape.com/article/1174503-overview 3. R&D Systems: http://www.rndsystems.com/Products/5868-ar/RelatedInformation 4. Hsueh et.al, 2012. Journal of Hematology & Oncology. http://www.jhoonline.org/content/5/1/17 5. www.nature.com/cddis/journal/v4/n10/full/cddis2013359a.html